The Phospholipid Flippase ATP8B1 is Involved in the Pathogenesis of Ulcerative Colitis via Establishment of Intestinal Barrier Function

Abstract Aims Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. Methods ATP8B1 expression was investigated in intestinal samples of patients with Crohn’s disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. Results ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. Conclusion ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.


Introduction
ATP8B1 is a phospholipid flippase belonging to the P4-ATPase family of proteins and it exerts its cellular functions by actively transporting phospholipids across cellular membranes. 1The ATP8B1 gene is expressed in liver and in several non-hepatic tissues, including the small and large intestine. 2Mutations in ATP8B1 lead to progressive familial intrahepatic cholestasis 1 [PFIC1], a chronic autosomal recessive liver disorder, which is characterized by impaired bile formation and progressive liver damage culminating in fibrosis and cirrhosis. 3Patients usually present in the first year of life with jaundice and pruritis. 4,53][14] The restored bile flow in post-liver transplant PFIC1 patients causes re-entry of high concentrations of bile salts into the intestine, which consequently aggravates the intestinal phenotype. 15Recently, two groups independently reported inflammation in the intestine of PFIC1 patients who had undergone a liver transplantation. 10,16The consequence of ATP8B1 dysfunction on intestinal homeostasis and inflammation is as yet unclear.Therefore, here we investigated the role of ATP8B1 in the intestine in vivo and in vitro as well as in relation to inflammatory bowel disease [IBD].
In this study, we show that ATP8B1 is highly expressed in epithelial cells of the intestine and is specifically reduced in patients with ulcerative colitis [UC] and in dextran sodium sulphate [DSS]-induced colitis in mice.We experimentally address the function of ATP8B1 in intestinal homeostasis by treating ATP8B1-deficient mice with DSS.ATP8B1-deficient mice displayed increased DSS sensitivity that was associated with increased intestinal permeability.Mechanistic studies in Caco2-BBE cells demonstrated an essential function for ATP8B1 in the establishment of epithelial barrier function.

Patient material
Fresh tissue samples were taken from IBD patients who underwent colorectal resection in the Amsterdam University Medical Center, location AMC.Normal colonic samples were taken from colorectal cancer patients at least 5 cm from the tumour.The study protocol was approved by the Medical Ethical Committee of the AMC [IBD Biobank, METC2014-178] and all patients provided written informed consent.Samples were immediately snap frozen and stored at −70°C until extraction, or fixed in 4% formaldehyde and routinely embedded in paraffin.Paraffin-embedded colorectal biopsies were obtained from a PFIC1 patient after liver transplantation that has been described by Kavallar

Mice
All experiments were approved by the Dutch government [Centale Commissie Dierproeven] under approval no.AVD1180020185344, and performed in accordance with national guidelines and regulations.Atp8b1 G308V/G308V /c57Bl/ J6 and wild-type c57Bl/J6 mice were bred in house and mice 6-10 weeks old of both genders were used, equally distributed over the groups.Mice were kept under specific pathogenfree conditions in individually ventilated cages.For induction of colitis drinking water was supplemented with

Mice colitis severity scores
The Disease Activity Index [DAI, score 0-9] was determined, consisting of severity of oedema [0-3], diarrhoea [0-3], and faecal occult blood [0-3].The length of the large intestine was measured in a relaxed position without stretching and weighed after removal of faeces.Colon density [mg/cm] was calculated by dividing the weight by the length of the large intestine.The intestine was opened longitudinally and washed thoroughly with phosphate-buffered saline [PBS], longitudinally divided in two, and one side was processed as a 'swiss role' into paraffin, the other side being immediately snap frozen and stored at −70°C.Slides of 5 µm were cut and stained with haematoxylin and eosin [H&E] using a standard protocol, and a standardized scoring system [score 0-12] was used to assess the severity of colitis consisting of two parameters: severity of inflammation [0-6]: 0 = none; 1 = infiltration in the lamina propria; 2 = confluence of inflammatory cells extending into the submucosa; 3 = transmural extension of the inflammatory infiltrate, combined with the extent of the inflammation [0 = none; 1 = <25%; 2 = 25-50%; 3 = >50%], and crypt damage [0-6]: 0 = intact crypts; 1 = loss of the basal half of the crypt; 2 = entire crypt loss; 3 = erosions/complete loss of epithelial layer, combined with extent [1 = <25%; 2 = 25-50%; 3 = >50%].

In situ hybridization
Sections of 5 µm were cut freshly and dried overnight at 37°C.In situ hybridization was performed according to the manufacturer's guidelines using the RNAscope Reagent 2.5HD Brown system [Advanced Cell Diagnostics] using the mmAtp8b1or hsATP8B1 probe [Bio-techne Ireland].

TEER and 4FD permeability assays
Transepithelial electrical resistance [TEER] was measured with a Millicell ERS-2 Voltohmeter [Millipore], according to the manufacturer's instructions.TEER values were corrected for background values [empty transwell] and surface area [0.33 cm 2 ].To evaluate the paracellular permeability of the Caco2-BBE monolayers, translocation of 4FD [Sigma-Aldrich] from the apical to basolateral side was measured.Therefore, 100 µL of 1 mg/mL 4FD in culture medium was added to the apical chamber and the amount of 4FD in the basolateral compartment was determined after 2 h using a CLARIOstar monochromator microplate reader [BMG Labtech].

Calcium switch assay
To study tight junction [TJ] assembly, TJs were disrupted by the Ca 2+ chelator EGTA [Sigma-Aldrich].Transwell monolayers cultured for 21 days were washed twice with calciumfree, magnesium-free EBSS [Eagle's balanced salt solution], before treatment with 2 mM EGTA in EBSS [both basolateral and apical] for 20 min.The monolayers were then washed once with EBSS and once with culture medium, and replaced by culture medium.TEER was measured before calciumswitch, immediately after and then every 2 h.

Immunofluorescent staining of cells
Transwell monolayers or cells grown on glass cover slips were washed with PBS and fixed with 4% paraformaledehyde for 10 min at RT.After thorough washings with PBS, cells were permeabilized with 0.1% Triton in PBS [PBS/Tx] and blocked in 5% goat serum [Dako] in 1% BSA/PBS and subsequently incubated with antibody to Claudin-4 [1:100] in PBS/Tx for 2 h at RT and washed with PBS/Tx.Cells were then incubated with goat-anti-rabbit-alexaFluor488 or alexaFluor594 [Invitrogen] for 1 h at RT, washed with PBS/Tx, and mounted with ProLong Gold Antifade reagent with DAPI [Thermo Fisher Scientific].Images were taken with a Leica DM6000 microscope using LAS AF software [Leica].For transverse sections, transwells were fixed with 4% paraformaldehyde for 30 min at RT and washed with PBS twice.After storing in 70% ethanol, transwells were cut from the chambers and imbedded in histogel [Thermo Scientific], embedded in paraffin and sectioned.CLDN4 immunofluorescent staining was done after antigen retrieval using Tris-EDTA pH 9.0 in a co-staining with goat-anti-GFP [1:100, Rockland, 600-101-215] to amplify the eGFP signal, and donkey-antirabbit-AlexaFluor546 [1:500, Invitrogen, A10040] and donkey-anti-goat-AlexaFLuor488 as secondary antibodies.

Proliferation assays
Cell proliferation was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay [Promega].In total, 2 × 10 3 cells were seeded in 100 µL per well in a 96-well plate and after 1, 2, 3, and 4 days 20 µL MTS solution was added and the absorbance was measured at 490 nm after 4 h.The absorbance values were corrected for the background [cell culture medium with MTS] values.Additionally, 1 × 10 3 cells were plated in six-well plates and cultured for up to 2 weeks.Cells were then fixed with 4% formaldehyde in PBS for 15 min and subsequently stained with 5 mg/mL crystal violet [C3886, Sigma-Aldrich] in 2% ethanol for 20 min.After thorough washings with H 2 O plates were air-dried and photographed.Subsequently stained cells were lysed in 200 µL lysis buffer [CST], 100 µL was transferred to a 96-well plate, and crystal violet absorbance was measured at 570 nm and corrected for blank [lysis buffer only] values.

Quantitative reverse-transcription PCR
RNA isolation and FACSorting of human and mouse colonic cell populations was as described previously. 20For cDNA synthesis equal concentrations of mRNA were

ATP8B1 is downregulated in UC
To investigate the expression of ATP8B1 in the human intestine we first analysed publicly available gene expression data sets, including ileal and colonic tissue samples from healthy control individuals.This revealed that ATP8B1 is highly expressed in the colon, when compared to the ileum [Supplementary Figure S1A].Analysis of single cell RNA sequencing [scRNAseq] data indicated that ATP8B1 is highly expressed by epithelial cells and endothelial cells in both the small intestine and colon [Supplementary Figure S1B].This was corroborated by qPCR on cell sorted populations from human colonic tissue [Supplementary Figure S1C].Immunohistochemistry indicated that ATP8B1 protein is mainly detected in the apical domain, as well as in lateral membranes of colonic intestinal epithelial cells, and confirmed complete absence of ATP8B1 protein in a colorectal biopsy of a PFIC1 patient [Figure 1A].Notably, the colorectal biopsy from the PFIC1 patient showed overt signs of chronic inflammation [Supplementary Figure S1D].Next, we sought to interrogate whether ATP8B1 expression was changed within the gastrointestinal tract in inflammatory conditions.Therefore, we first analysed published transcriptomic data of intestinal biopsies from two different cohorts of IBD patients [GSE16879, GSE75214], including CD and UC.As ATP8B1 expression is highest in the colon, we focused on the colonic expression as this also enabled us to compare CD with UC.ATP8B1 expression was significantly decreased in both UC patient cohorts, while only one cohort showed a significant decrease of ATP8B1 expression in CD [Figure 1B].Further analysis of both cohorts showed that decreased ATP8B1 expression was specifically associated with active UC when compared to non-active UC [Figure 1C].In line with this, ATP8B1 was increased upon disease treatment [Figure 1C, 'before versus after'].Analysis of additional patient cohorts confirmed that ATP8B1 expression was decreased in diseased intestinal tissue of UC patients [Figure 1D].In situ hybridization as well as immunohistochemical staining showed decreased ATP8B1 levels in UC compared to healthy colon tissue [Figure 1E].Immunoblotting of intestinal colon samples indeed confirmed ATP8B1 protein levels being reduced in UC compared to healthy controls [Figure 1F, G].Villin levels were not affected in UC samples, indicating that the decreased ATP8B1 levels were not simply a reflection of loss of epithelial cells during ulcerating conditions [Figure 1F, G].

Atp8b1 expression in experimental mouse models of colitis
Next, we investigated intestinal expression of Atp8b1 in several murine models of IBD.Similar to humans, mice expressed higher levels of Atp8b1 in the colon compared to ileum [Figure 2A].scRNAseq revealed that in mice Atp8b1 is highly expressed in epithelial cells, but also in fibroblasts and cells of the lymphatic system [Supplementary Figure S2A, B

DSS induces severe colitis in ATP8B1deficient mice
To examine if loss of ATP8B1 in the intestine affects colonic inflammation, we challenged Atp8b1 G308V/G308V mutant mice with DSS.Atp8b1 G308V/G308V mutant mice [hereafter denoted Atp8b1-deficient mice] are knock-in mice for the prototypic PFIC1 mutation from the Byler family, 3 a glycine to valine substitution at position 308 [G308V], that results in near absence of the protein.

ATP8B1 is involved in intestinal epithelial barrier function
To identify molecular pathways that correlated with reduced intestinal ATP8B1 expression in UC patients, we investigated gene expression associations in an unbiased manner by splitting intestinal UC samples into two groups according to their ATP8B1 expression [i.e.above and below the median ATP8B1 expression] in a patient cohort [GSE109142, n = 206].Gene set enrichment analysis [GSEA] revealed a close correlation between TJ pathway-associated gene expressions and ATP8B1 expression [Figure 4A], suggesting that ATP8B1 deficiency is associated with a defect in intestinal barrier function.
To investigate whether enhanced sensitivity to DSS colitis in Atp8b1-deficient mice is associated with a diminished epithelial barrier function, Atp8b1-deficient mice were challenged for 3 days with 2% DSS.This allowed us to assess the epithelial barrier integrity in vivo upon the initial epithelial insult, but before the manifestation of a severe colitis.After 3 days of DSS treatment there were no signs of body weight loss in either wild-type or Atp8b1-deficient mice [Supplementary Figure S3].Intestinal permeability, as assessed after oral gavage of 4FD, was not affected in water-or DSS-treated wild-type mice nor in water control Atp8b1-deficient mice, indicating that barrier function was not impaired 3 days post-DSS, nor in naïve Atp8b1deficient mice [ differentiation or proliferation as neither Villin levels nor cellular proliferation rates were affected [Figure 5B, C and Supplementary Figures S4 and S5].
To accelerate attaining confluence and barrier function, we seeded a higher cell density in the transwell filters.Under these conditions, and as reported by others, the TJs are assembled within 4 days. 23Notably, during the first 7 days, TEER values of ATP8B1 KD cells lagged behind control cells by ~50% [Figure 5D], a phenotype that coincided with a 4-fold increased passage of 4FD [Figure 5E].Despite the lag in initial TEER development, resistance was completely recovered after 21 days of culture, suggesting a role for ATP8B1 in the initial phase of barrier establishment.We therefore performed a calcium switch assay, where we disrupted the 21-day fully differentiated TJ complexes after which we monitored restoration of barrier function.Ca 2+ withdrawal resulted in an ~85% drop in TEER, which was equal in both cell lines [Figure 5F and Supplementary Figure S6].After Ca 2+ re-addition, ATP8B1 KD cells displayed a significantly reduced recovery of TEER, suggesting a delayed barrier reassembly [Figure 5F].The delayed development of TEER coincided with an ~3-fold increase in permeability to 4FD relative to short-hairpin control (shControl) cells [Figure 5G].Altogether, these data indicate a crucial role for ATP8B1 in the initial establishment of the epithelial barrier.

ATP8B1 and Claudin-4 have complementary functions in barrier function in Caco-2 cells
To investigate the molecular mechanisms involved in ATP8B1associated barrier establishment, we assessed the levels of several TJ proteins on day 7 transwell inserts.ATP8B1 KD cells displayed no/little changes in multiple TJ protein levels, except for CLDN4 levels, which were slightly elevated [Figure 6A, B, Supplementary Figure S7].Since elevated CLDN4 levels have been associated with increased rather than impaired barrier function, 24 we investigated the cellular localization of CLDN4 in the ATP8B1 KD monolayers.Immunofluorescence staining revealed that ATP8B1 KD cells showed a diffuse cytoplasmic staining pattern for CLDN4, as opposed to a membraneassociated CLDN4 staining in control cells [Figure 6C].These data suggest that in ATP8B1 KD cells, CLDN4 is not properly localized and as a consequence accumulated in these cells.
To assess a possible interaction between ATP8B1 and CLDN4 we studied their localization in ATP8B1-eGFP over-expressing Caco2-BBE cells.The ATP8B1-eGFP fusion is functional, 19 and its over-expression did not influence epithelial barrier function, as judged by TEER and 4FD permeability measurements [Supplementary Figure S8].In Caco2-BBE cells grown in a monolayer ATP8B1-eGFP, as previously shown, 19 localized to the plasma membrane and co-stained with CLDN4 [Figure 6D], suggesting that both proteins localized to or near the plasma membrane.Translateral slides of Caco2-BBE-ATP8B1-eGFP cells grown on transwells for 7 days confirmed apical membrane staining of ATP8B1-eGFP and showed a punctate enrichment for CLDN4, reminiscent of tight junctional staining [Figure 6E].To assess whether the two proteins could physically interact, we co-immunoprecipitated ATP8B1-eGFP from the cells and eluted a small fraction of CLDN4, suggesting that ATP8B1 could interact with CLDN4 [Figure 6F]; this interaction was specific for CLDN4 since ATP8B1-eGFP did not precipitate with GAPDH.Furthermore, ATP8B1 was clearly attached to the cytoskeleleton, indicated by β-actin co-immunoprecipitation.These data suggest strongly that a small fraction of the cellular CLDN4 pool specifically interacts with ATP8B1.To establish the contribution of CLDN4 to barrier formation, we generated CLDN4 KD Caco2-BBE cells.Knockdown led to a strong decrease in cellular proliferation [Supplementary Figure S9] which hampered measurement of the epithelial barrier by TEER and 4FD translocation.Immunoblot analyses showed that in CLDN4 KD cells, ATP8B1 levels were reduced [Figure 6G, H].Although we cannot exclude a secondary consequence of affected proliferation, these data suggest an inter-dependence of the two proteins.Similar to control Caco2-BBE cells, immunohistochemical staining of CLDN4 in human healthy control colon showed clear tight junctional, as well as lateral, staining in epithelial cells [Figure 7C].We found that tight junctional staining of CLDN4 was less pronounced or even absent in intestinal samples from UC patients whereas lateral staining was unaffected [Figure 7A].This correlated with decreased ATP8B1 protein staining in the same UC samples [Figure 1A, E].Importantly, the colonic epithelium of the ATP8B1-deficient [PFIC1] patient virtually lacked tight junctional signal for CLDN4 [Figure 7A].
In Atp8b1-deficient mice, both under control conditions and after 3 days of DSS exposure, intestinal CLDN4 levels were similar to those in wild-type mice [Figure 7B, C], which decreased dramatically after 6 days of DSS exposure in both wild-type and Apt8b1-deficient mice [Figure 7D,  E].Altogether, these data underline an important role for ATP8B1 in conjunction with CLDN4 in tight junctional integrity in intestinal epithelial cells.

Discussion
In the present study we provide evidence for an important role for the phospholipid flippase ATP8B1 in the pathogenesis of UC via the establishment of barrier function in intestinal epithelial cells.First, we show that ATP8B1 was highly expressed in colonic intestinal epithelial cells and was down-regulated in colon tissue of patients with UC.Similarly, we show downregulation of colonic Atp8b1 in two mouse models of UC-like IBD.Second, Atp8b1-deficient mice were extremely sensitive to DSS exposure as indicated by the induction of wasting disease 6 days post-DSS, while wild-type mice showed only symptoms of mild disease.Third, we show that ATP8B1 fulfils a function in the establishment of the epithelial barrier both in vivo and in vitro.Furthermore, we show that ATP8B1 interacted with a small fraction of the cellular CLDN4 pool in Caco2-BBE cells.Importantly, CLDN4 was mislocalized in a colorectal biopsy of an ATP8B1-deficient [PFIC1] patient, while both ATP8B1 and CLDN4 were down-regulated and/ or mislocalized in UC samples.Altogether, these observations suggest strongly that down-regulation of ATP8B1 can be an initiating trigger to impair intestinal barrier function, possibly via impairment of TJ integrity, with consequent aggravation of intestinal inflammation.Although our data show that barrier dysfunction in ATP8B1 low/deficient conditions is associated with CLDN4 mistargeting, we cannot rule out a possible contribution of other TJ proteins in this phenotype.
Our data indicate that ATP8B1 is crucially involved in the establishment, rather than in the homeostatic maintenance, of the intestinal epithelial barrier and that a 'second hit' is required to expose or aggravate an intestinal phenotype.This is evidenced by two observations in vivo and in vitro.First, Atp8b1-deficient mice do not display an impaired intestinal epithelial barrier and lack an overt intestinal phenotype.During a DSS-induced intestinal insult ['second hit'], however, barrier dysfunction was observed even at 3 days post-DSS in Atp8b1-deficient mice, but not in wild-type mice, which progressed to severe epithelial damage and aggravating colitis 6 days after DSS.Second, ATP8B1 KD Caco-2-BBE cells displayed delayed barrier formation in the first 14 days of differentiation in a transwell culture, whereas barrier function was completely restored to that of control cells in fully differentiated cells.However, when a 'second hit' was employed on fully differentiated cells, i.e. barrier disruption by calcium withdrawal, re-establishment of the barrier was strongly impaired, indicating a role for ATP8B1 in barrier establishment.
The 'second hit' hypothesis could also explain the intestinal phenotype in post-liver transplant [LT] ATP8B1-deficient PFIC1 patients.Apart from severe cholestatic liver disease, extrahepatic phenotypes, including diarrhoea, are common in PFIC1 patients. 4,7,9,25After liver transplantation, however, >80% of PFIC1 patients continue to develop exacerbated diarrhoea, [26][27][28] which is probably due to restored bile flow into the intestine.0][31] We propose that increased bile salt entry into the intestine of post-LT PFIC1 patients serves a 'second hit' that damages the intestinal epithelium, causing epithelial barrier defects and consequent diarrhoea and inflammation, and triggers hepatic steatosis.Importantly, Stephanie and colleagues reported a mild and chronic inflammation in intestinal biopsies from a post-LT PFIC1 patient, 16 while Henkel and colleagues recently reported on a post-LT PFIC1 patient who had developed colitis specifically in the distal colon. 10Hence, our observations indicate an essential function for ATP8B1 in the establishment of the intestinal barrier, and suggest strongly that a 'second hit' is required to expose a barrier defect as well as colitis when ATP8B1 levels are reduced/absent.
We find that both ATP8B1 and CLDN4 are involved in the epithelial barrier phenotype in Caco2-BBE cells.3][34] With their extracellular domains they form paracellular seals, while in most claudins the intracellular C-terminal domains interact with other, PDZdomain containing TJ proteins, signalling molecules, as well as with the actin network. 32,35,36In Caco2-BBE cells, we find a strong link between ATP8B1 and CLDN4.First, ATP8B1 KD Caco2-BBE cells showed reduced TEER build-up during the first 7 days in a transwell culture as well as elevated total CLDN4 levels.Although previous research showed that transgenic over-expression of CLDN4 resulted in increased TEER in MDCK cells, 37 we hypothesize that the elevated CLDN4 levels in ATP8B1 KD cells are a consequence of mis-targeting and consequent cellular accumulation of CLDN4.Second, ATP8B1-eGFP and CLDN4 co-stained in the plasma membrane and co-immunoprecipitated, indicating that they physically interact.Our co-immunoprecipitation data indicate that only a small fraction of total CLDN4 precipitated with ATP8B1, which can be caused by either a low-affinity interaction between the two proteins or that just a small fraction of the CLDN4 pool is bound to ATP8B1.Collectively, our observations indicate that ATP8B1 has an important role in the establishment of barrier function, possibly via the maintenance of TJ integrity, in which CLDN4 plays an important role.
From our data we hypothesize that ATP8B1 is involved in the targeting of CLDN4, and possibly other yet to be identified proteins, to the TJ.9][40] Although our finding that elevated CLDN4 levels in ATP8B1 KD cells contrasts with the reduced CLDN4 levels found in UC patients, 38,41,42 it is possible that the initial mislocalization [as observed in Caco2-BBE cells] causes the protein to accumulate, whereas under progressive inflammatory conditions, proteins, including CLDN4, will be degraded, decreasing total CLDN4 levels.
We have identified ATP8B1 as an essential protein in intestinal barrier establishment, and a novel, possible early-stage, determinant of UC pathogenesis.

Figure 1 .
Figure 1.ATP8B1 expression in UC. [A] ATP8B1 expression in the human intestine as determined by immunohistochemistry on colonic tissue of a healthy control [HC] and a ATPB1-deficient PFIC1 patient.[B] Analysis of human ATP8B1 expression in HC and UC and CD colonic biopsies.Data were derived from GSE75214 [HC, n = 11; CD, n = 8; UC, n = 97] and GSE16879 [HC, n = 6; CD, n = 37; UC, n = 78].[C] ATP8B1 expression in active [n = 74] ]. Upon induction of intestinal inflammation with DSS, an experimental IBD model that resembles UC, a decrease in Atp8b1 expression was detected [Figure 2B].Additionally, in IL10 knock-out mice, that spontaneously develop [UC-like] intestinal inflammation, a reduction in intestinal Atp8b1 expression was overt [Figure 2B].In contrast, in intestinal inflammation of the T-cell transfer model, which is considered more CD-like, intestinal Atp8b1 expression was unaffected [Figure 2B].In situ hybridization confirmed the reduction of epithelial Atp8b1 in DSS-induced colonic inflammation [Figure 2C] and this coincided with reduced ATP8B1 protein levels [Figure 2D], also when corrected for epithelial cell abundance by Villin levels [Figure 2D].
17d colleagues under IRB approval of the Medical University of Innsbruck [EK 1029/2017].17 2.0% [w/v] DSS [mol.wt 36 000-50 000 kDa; Sigma Aldrich], provided ad libitum and refreshed every other day.Body weights were measured daily during DSS administration.Mice were killed by CO 2 suffocation and the large intestine was removed.All measurements were performed in a blinded fashion [see Supplementary Material & Methods].To determine intestinal permeability in vivo, mice were starved for 4 h after which FITC-conjugated dextran [4 kDa, 4FD; Sigma-Aldrich] was administered by oral gavage [60 mg/100 g body weight].After 3 h, mice were killed and blood was collected by cardiac puncture.Plasma samples were measured for FITC fluorescence intensity [ex/em = 485/520 nm] using a CLARIOstar monochromator microplate reader [BMG Labtech].
21verse transcribed using oligo[dT], random hexamers, and M-MuLV reverse transcriptase [RT] from the First Strand cDNA Synthesis kit [Thermo Scientific] according to the manufacturer's protocol.Quantitative RT-PCR [qPCR] was performed using a sensifast SYBR No-ROX Kit [Bioline] on a BioRad iCycler.GeneNorm was used to select multiple stable housekeeping genes.Analysis was performed by using the LinReg method.21Primersequences to human ATP8B1: